Intercellular communication is essential for the function of multicellular systems. Ion channel proteins, as mediators of information transfer in the brain, endocrine system, enteric nervous system and neuromuscular junction, modulate ion fluxes that produce voltage changes across cell membranes and simultaneously act as sensors of physiological signals, for example, changes in ligand concentrations and in transmembrane voltage. Ligand-gated ion channels provide for rapid dialogue between cells of the central nervous system, converting a chemical neurotransmitter signal released from one cell into an electrical signal that propagates along the cell membrane of a target cell. Ligand-gated ion channels are multimeric protein complexes with component subunits encoded by related genes.
At the present time, numerous families of ligand-gated receptors have been identified and characterized on the basis of sequence identity. Those which form cationic channels include, for example, excitatory nicotinic acetylcholine receptors (nAChRs), excitatory glutamate-activated receptors, the 5-HT3 serotonin receptor, the ATP receptor and the sarcoplasmic ryanodine receptor. Those which form anionic channels include, for example, the inhibitory GABA and glycine-activated receptors.
The neurotransmitter acetylcholine (ACh) activates two pharmacologically different receptor types: nicotinic acetylcholine receptors (nAChR) from the ligand-gated ion channel superfamily and muscarinic acetylcholine receptors (mAChR) from the G-protein coupled receptor superfamily (Taylor, A. Goodman-Gilman, T. H. Rall, A. S. Nies and P. Taylor, eds. (New York:Pergamon Press), pp. 166-186,1990); (Taylor, A. Goodman-Gilman, T. H. Rall, A. S. Nies and P. Taylor, eds. (New York:Pergamon Press), pp. 122-149,1990). A number of pathologies and/or disease conditions are associated with nAChRs, such as, for example, myasthenia gravis, schizophrenia, Alzheimer""s disease, Tourette""s disease and nicotine addiction. Biochemical and electrophysiological data have shown that nicotinic and muscarinic receptors are functionally distinct entities. (Bonner, et al., Science, 237, 527-532, 1987). Whereas nAChRs are pentamers composed of related protein subunits that span the plasma membrane four times, mAChRs are formed by a single polypeptide chain which is postulated to span the plasma membrane seven times.
Nicotinic acetylcholine receptors, glycoproteins composed of five subunits, transduce the binding of acetylcholine in the cationic channel. The five receptor subunits form a pseudosymmetric ring around a central channel. Neuronal nicotinic AChRs (NnAChRs) mediate neurotransmission at many central and peripheral synapses, and comprise two subunit types (alpha and beta) encoded by 10 different neuronal genes. Expression of particular combinations of subunit RNAs in oocytes yields biophysically distinct channels that are distinguished pharmacologically on the basis of ligands that modulate such channels.
Recombinant DNA technology has enabled the identification of the vertebrate muscle nAChR subunits alpha1, beta1, gamma, delta and epsilon and the neuronal subunits alpha2, alpha3, alpha4, alpha5, alpha6, alpha7, alpha8, beta2, beta3 and beta4 (rat nomenclature). Various combinations of these subunits produce functional recombinant receptor-channel complexes that are activated by both ACh and nicotine. The nAChR at the neuromuscular junction is thought to have a (xcex11)2xcex21xcex3xcex4 stoichiometry (Galzi, et al., Annu. Rev. Pharmacol., 31, 37-72, 1991). In contrast, the neuronal nAChR subunits alpha2, alpha3 and alpha4 lead to the assembly of functional nAChRs in concert with either beta2 or beta4 (Boulter, et al. Proc. Natl. Acad. Sci. USA, 84, 7763-7767, 1987; Ballivet, et al., Neuron, 1, 847-852, 1988; Wada, et al., Science, 240, 330-334, 1988; Deneris, et al., Neuron, 1, 45-54, 1988; Duvoisin, et al., Neuron, 3, 487-496, 1989; Couturier, et al., J. Biol. Chem, 265, 17560-17567, 1990), while the neuronal alpha7 and alpha8 subunits can form functional nAChRs in the absence of any other subunit (Couturier, et al., J. Biol. Chem, 265, 17560-17567, 1990; Seguela, et al., J. Neurosci, 13, 596-604, 1993; Gerzanich, et al., Molec. Pharmacol., 45, 212-220, 1994).
Given the existence of ten distinct nicotinic acetylcholine subunit genes, numerous combinations of subunits producing functional receptors are possible. In spite of the numerous combinations of subunits which can be prepared from previously cloned genes, the properties of the native nAChRs do not always match those of recombinant receptors (Sargent, Annu. Rev. Neurosci., 16, 403-443, 1993). For example, the cholinergic receptors present in bovine chromaffin cells and in rat and chick cochlear hair cells exhibit a pharmacological profile that does not fit any combination of known subunits (Shirvan, et al., Proc. Natl. Acad. Sci. USA., 88, 4860-4864, 1991; Housley, et al., Proc. R. Soc. Lond. B, 244, 161-167, 1991; Fuchs, et al., Proc. R. Soc. Lond. B, 248, 35-40, 1992; Erostegui, et al., Hearing Res., 74, 135-147, 1994), thus suggesting the existence of additional, as yet unidentified subunits.
Thus, a need exists for identifying additional members of the nicotinic acetylcholine receptor superfamily, and characterizing such nAChR subunits, as well as functional receptors assembled therefrom, which includes elucidation of the nature of assembly of various subunits in the production of a functional receptor (i.e., a subunit assembly containing ligand binding sites and a ligand-gated transmembrane channel), and the relationship between the structure of the subunit assembly and the pharmacological profile of the corresponding receptor. The present invention satisfies these needs and provides related advantages as well.
The present invention provides isolated nucleic acids encoding alpha9 nicotinic acetylcholine receptor (nAChR) subunit, isolated receptor subunit protein encoded thereby as well as recombinately expressed alpha9 nicotinic acetylcholine receptor (nAChR). Further provided are vectors and probes containing such nucleic acids, host cells transformed with such nucleic acids, antisense oligonucleotides and compositions containing such oligonucleotides, antibodies that specifically bind to invention receptors and compositions containing such antibodies as well as transgenic non-human mammals.
The alpha9 nAChR subunits of the invention form a cationic receptor channel complex which is activated by acetylcholine and is permeable to cations, including calcium. Functional alpha9 nACh receptors of the invention may be expressed as homomeric receptors, i.e., only one type of subunit is required for function, or invention receptors may be expressed as heteromeric receptors wherein more than one type of subunit is required to form a functional receptor. Additionally, the invention provides methods for identifying compounds that modulate activity of the invention receptors, or the activity of nucleic acid encoding such receptors.